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1 Lipid Rafts, Caveolae, and Membrane Traffic Doris Meder and Kai Simons

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1 Lipid Rafts, Caveolae, and Membrane Traffic Doris Meder and Kai Simons 1 1 Lipid Rafts, Caveolae, and Membrane Traffic Doris Meder and Kai Simons 1.1 Introduction Cell membranes are dynamic assemblies of a variety of lipids and proteins. They form a protective layer around the cell, but also mediate the communication with the outside world – that is, neighboring cells in a tissue, hormones and growth factors arriving with the blood supply, or pathogens trying to enter the system. The unique feature of cell membranes is that their lipid and protein constituents can self-assemble into 5 nm-thin, two-dimensional fluids composed of two apposing lipid monolayers that form a hydrophobic interior and two polar interfacial regions oriented towards the aqueous medium. This organizing principle – the lipid bi- layer – is the oldest, still valid molecular model of biological structures. The first model that incorporated proteins was proposed by Danielli and Davson, and as- sumed that the bilayer was made up entirely of lipids and that proteins covered the two polar surfaces [1]. Some 40 years later, the fluid mosaic model of the cell membrane proposed by Singer and Nicolson [2] was a conceptual breakthrough. Amphipathic membrane proteins were recognized to reside within, and even span, the whole bilayer that was depicted as a dynamic structure, the components of which are laterally mobile. However, the view that the lipids in the bilayer mainly serve as a homogeneous solvent for proteins [2] has been proven to be too simplistic. Lipids are not only distributed asymmetrically between the two leaflets of the bilayer, but also within the leaflet they are heterogeneously arranged [3]. This chapter will recapitulate the history and recent advances in membrane biology including the lipid raft concept, and then summarize current views on the func- tions of rafts and caveolae in membrane traffic. 1.2 Basic Organization Principles of a Cell Membrane The lipid bilayer is a two-dimensional fluid, where lipid molecules exchange slowly between leaflets but are mobile within the leaflet. This mobility consists of two parts: Lipid Rafts and Caveolae. Christopher J. Fielding Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31261-7 2 1 Lipid Rafts, Caveolae, and Membrane Traffic • the “translational freedom” of a molecule – that is, its lateral mobility; and • the “configurational freedom” that is, the ability to flex parts of the molecule and to rotate bonds in its carbon backbone. Synthetic bilayers change from a liquid state with high translational and configura- tional freedom into a rigid gel state at a characteristic freezing point. Cell mem- branes at physiological temperatures are almost always in the liquid state, but can contain regions with high configurational order, as will be described later. Im- portantly, the lipid bilayer of cell membranes is asymmetric, with a different lipid composition in the two leaflets. The main lipid components of cellular membranes are glycerophospholipids, with the most abundant species being phosphatidylcho- line (PC) in the exoplasmic leaflet and phosphatidylethanolamine (PE) and phos- phatidylserine (PS) in the inner leaflet, as well as sphingolipids with glycosphingo- lipids and sphingomyelin (SM) mostly localized to the exoplasmic leaflet. Sterols make up the third lipid class, and are present in both leaflets. Mammalian cell membranes contain only one sterol, namely cholesterol, but probably more than thousand different glyco- and sphingolipid species, emerging from the combinato- rial propensity to assemble lipids from different backbones linked in different ways with two varying hydrocarbon chains and a vast number of headgroups. A large number of flippases and translocators tightly control the asymmetric dis- tribution of all these lipids across the bilayer [4]. Lipids are differentially distributed between cellular organelles. The endoplas- mic reticulum and the Golgi-complex contain mainly glycerophospholipids and only small amounts of sphingolipids, whereas the plasma membrane is relatively enriched in SM and glycosphingolipids [5]. Also within the membrane plane of one organelle, lipids are believed to be heterogeneously arranged. Caveolae – small invaginations of the plasma membrane – are enriched in glycosphingolipids [6], and phosphatidylinositol-3’-phosphate (PI(3)P) is concentrated in subdomains of early endosome membranes [7]. Recently, vacuole-fusion in yeast has been shown to be controlled by microdomains of ergosterol, diacylglycerol and phosphoinosi- tide-3-and-4-phosphate [8]. Furthermore, membranes are differentially susceptible to extraction by detergents such as Triton X-100 or CHAPS at 4 °C, with some proteins and lipids being completely solubilized and others forming so-called “de- tergent-resistant membranes” (DRM; for a review, see [9]). These findings sug- gested that cell membranes contained microdomains in which lipids were more tightly packed and thus not accessible to the detergent, although it is widely ac- cepted that DRMs do not have an exact in-vivo correlate but are defined by being formed during the detergent treatment [10]. These microdomains were later termed “rafts” and were described as sphingolipid-cholesterol assemblies contain- ing a subset of membrane proteins [11]. Currently, the raft hypothesis is heavily debated [12–14], with the main discussion points being the methodologies to study rafts and the size of the domains (see below). The core of the raft concept is that cell membranes phase-separate into different domains and that this is a lipid- driven process. In light of the ongoing discussion in the field, the following sec- tions will provide an overview about what is known about phase separation, first 1.3 Evidence for Phase Separation in Model Membrane Systems 3 discussing the studies conducted in model membrane systems and later in cell membranes. 1.3 Evidence for Phase Separation in Model Membrane Systems: Liquid-Ordered and Liquid-Disordered Phases Various model membrane systems have been used by physicists and chemists to study phase separation in lipid mixtures. They are either monolayers or bilayers. Monolayers are either assembled at an air-water interface with the packing density of the lipids being adjusted by applying lateral pressure, or on a supporting lipid monolayer that is fixed to a solid support. Bilayers are used in the supported ver- sion as described above, or in the form of vesicles. The most commonly used vesicles are large or giant unilamellar vesicles (LUV or GUV, respectively) com- posed of only a single bilayer, but also multilamellar vesicles (MLV) are used. The basic principles were first established in simple binary lipid mixtures, but recently ternary mixtures which more closely mimic the composition of the cell plasma membrane have been used. The mixtures usually contain one lipid with a high melting temperature (Tm), one with a low Tm, and cholesterol. GUVs are probably the system closest to a cell membrane, because artifacts from a support are ex- cluded. Still, cell membranes are asymmetric with different lipid compositions of the outer versus the inner leaflet, while the GUVs used so far were all symmetric. Since maintaining an asymmetric lipid distribution is energy-consuming, perhaps by reconstituting lipid translocators into liposomes this drawback can be overcome in the future. Although model membrane systems produce very simplified pic- tures of cell membranes, there are many examples of a close correlation with experimental data obtained in living cells [14]. Ipsen et al. were the first to describe the formation of a liquid-ordered phase by cholesterol and saturated phospholipids [15,16]. This phase can coexist with other lipid phases, and its characteristics are described as follows: the translational order of lipid molecules within the liquid-ordered phase is similar to that in a fluid bilayer state, whereas the configurational order of the hydrocarbon chains com- pares more to that in a gel state. The formation of the liquid-ordered phase was attributed to the unique chemical nature of cholesterol (for a review, see [17]), but later it was shown that all natural sterols promote domain formation and that also small amounts of ceramide (3%) can stabilize domains formed in vesicles [18]. Leventis and Silvius showed that the interaction of cholesterol with different lipid species is dependent on the nature of their hydrocarbon chains and, to a lesser extent, also on their headgroup. The interaction preference decreases with SM > PS > PC > PE and with increasing unsaturation of the acyl chains [19]. Whereas the kink in unsaturated hydrocarbon chains is likely to hinder tight pack- ing with the flat sterol ring of cholesterol, the reason for the preferential inter- action of cholesterol with SM is still a debated issue. The first visualization of “raft-like domains” in model membranes was achieved 4 1 Lipid Rafts, Caveolae, and Membrane Traffic by Dietrich et al. [20]. They visualized liquid ordered domains in supported bi- layers and GUVs composed not only of synthetic lipid mixtures but also of lipid extracts from brush border membrane, the apical membrane of intestinal cells. Domain formation was cholesterol-dependent, since domains disappeared after treatment with the cholesterol-extracting drug methyl-b-cyclodextrin. Another big step forward was the establishment of a ternary phase diagram of SM/PC/choles- terol at the physiological temperature of 37 °C [21]. This predicts the coexistence of liquid-ordered and liquid-disordered phases for a wide range
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